Marjorie F. Oleksiak

Variation in gene expression within and among natural populations

Evolution may depend more strongly on variation in gene expression than on differences between variant forms of proteins. Regions of DNA that affect gene expression are highly variable, containing 0.6% polymorphic sites. These naturally occurring polymorphic nucleotides can alter in vivo transcription rates. Thus, one might expect substantial variation in gene expression between individuals. But the natural variation in mRNA expression for a large number of genes has not been measured. Here we report microarray studies addressing the variation in gene expression within and between natural populations of teleost fish of the genus Fundulus. We observed statistically significant differences in expression between individuals within the same population for approximately 18% of 907 genes. Expression typically differed by a factor of 1.5, and often more than 2.0. Differences between populations increased the variation. Much of the variation between populations was a positive function of the variation within populations and thus is most parsimoniously described as random. Some genes showed unexpected patterns of expression—changes unrelated to evolutionary distance. These data suggest that substantial natural variation exists in gene expression and that this quantitative variation is important in evolution.

Natural variation in cardiac metabolism and gene expression in Fundulus heteroclitus

Individual variation in gene expression is important for evolutionary adaptation and susceptibility to diseases and pathologies. In this study, we address the functional importance of this variation by comparing cardiac metabolism to patterns of mRNA expression using microarrays. There is extensive variation in both cardiac metabolism and the expression of metabolic genes among individuals of the teleost fish Fundulus heteroclitus from natural outbred populations raised in a common environment: metabolism differed among individuals by a factor of more than 2, and expression levels of 94% of genes were significantly different (P < 0.01) between individuals in a population. This unexpectedly high variation in metabolic gene expression explains much of the variation in metabolism, suggesting that it is biologically relevant. The patterns of gene expression that are most important in explaining cardiac metabolism differ between groups of individuals. Apparently, the variation in metabolism seems to be related to different patterns of gene expression in the different groups of individuals. The magnitude of differences in gene expression in these groups is not important; large changes in expression have no greater predictive value than small changes. These data suggest that variation in physiological performance is related to the subtle variation in gene expression and that this relationship differs among individuals.

The genomic landscape of rapid repeated evolutionary adaptation to toxic pollution in wild fish

Mapping genetic adaptations to pollution Many organisms have evolved tolerance to natural and human-generated toxins. Reid et al. performed a genomic analysis of killifish, geographically separate and independent populations of which have adapted recently to severe pollution (see the Perspective by Tobler and Culumber). Sequencing multiple sensitive and resistant populations revealed signals of selective sweeps for variants that may confer tolerance to toxins, some of which were shared between resistant populations. Thus, high genetic diversity in killifish seems to allow selection to act on existing variation, driving rapid adaptation to selective forces such as pollution. Science, this issue p. 1305; see also p. 1232 Genetic diversity in Atlantic killifish has allowed for convergent evolution of pollution tolerance. Atlantic killifish populations have rapidly adapted to normally lethal levels of pollution in four urban estuaries. Through analysis of 384 whole killifish genome sequences and comparative transcriptomics in four pairs of sensitive and tolerant populations, we identify the aryl hydrocarbon receptor–based signaling pathway as a shared target of selection. This suggests evolutionary constraint on adaptive solutions to complex toxicant mixtures at each site. However, distinct molecular variants apparently contribute to adaptive pathway modification among tolerant populations. Selection also targets other toxicity-mediating genes and genes of connected signaling pathways; this indicates complex tolerance phenotypes and potentially compensatory adaptations. Molecular changes are consistent with selection on standing genetic variation. In killifish, high nucleotide diversity has likely been a crucial substrate for selective sweeps to propel rapid adaptation.

Signatures of selection in natural populations adapted to chronic pollution.

BackgroundPopulations of the teleost fish Fundulus heteroclitus appear to flourish in heavily polluted and geographically separated Superfund sites. Populations from three Superfund sites (New Bedford Harbor, MA, Newark Bay, NJ, and Elizabeth River, VA) have independently evolved adaptive resistance to chemical pollutants. In these polluted populations, natural selection likely has altered allele frequencies of loci that affect fitness or that are linked to these loci. The aim of this study was to identify loci that exhibit non-neutral behavior in the F. heteroclitus genome in polluted populations versus clean reference populations.ResultsTo detect signatures of natural selection and thus identify genetic bases for adaptation to anthropogenic stressors, we examined allele frequencies for many hundreds of amplified fragment length polymorphism markers among populations of F. heteroclitus. Specifically, we contrasted populations from three Superfund sites (New Bedford Harbor, MA, Newark Bay, NJ, and Elizabeth River, VA) to clean reference populations flanking the polluted sites. When empirical FST values were compared to a simulated distribution of FST values, 24 distinct outlier loci were identified among pairwise comparisons of pollutant impacted F. heteroclitus populations and both surrounding reference populations. Upon removal of all outlier loci, there was a strong correlation (R2 = 0.79, p < 0.0001) between genetic and geographical distance. This apparently neutral evolutionary pattern was not evident when outlier loci were included (R2 = 0.092, p = 0.0721). Two outlier loci were shared between New Bedford Harbor and Elizabeth River populations, and two different loci were shared between Newark Bay and Elizabeth River populations.ConclusionIn total, 1% to 6% of loci are implicated as being under selection or linked to areas of the genome under selection in three F. heteroclitus populations that reside in polluted estuaries. Shared loci among polluted sites indicate that selection may be acting on multiple loci involved in adaptation, and loci shared between polluted sites potentially are involved in a generalized adaptive response.

The biological importance of measuring individual variation

SUMMARY Functional genomics research using Fundulus heteroclitus has focused on variation among individuals because of the evolutionary importance and value of Fundulus in explaining the human condition (why individual humans are different and are affected differently by stress, disease and drugs). Among different populations and species of Fundulus, there are evolutionarily adaptive differences in gene expression. This natural variation in gene expression seems to affect cardiac metabolism because up to 81% of the variation in glucose utilization observed in isolated heart ventricles is related to specific patterns of gene expression. The surprising result from this research is that among different groups of individuals, the expression of mRNA from different metabolic pathways explains substrate-specific metabolism. For example, variation in oxidative phosphorylation mRNAs explains glucose metabolism for one group of individuals but expression of glucose metabolism genes explains this metabolism in a different group of individuals. This variation among individuals has important implications for studies using inbred strains: conclusions based on one individual or one strain will not necessarily reflect a generalized conclusion for a population or species. Finally, there are surprisingly strong positive and negative correlations among metabolic genes, both within and between pathways. These data suggest that measures of mRNA expression are meaningful, yet there is a complexity in how gene expression is related to physiological processes.

Convergence and divergence in gene expression among natural populations exposed to pollution

BackgroundNatural populations of the teleost fish Fundulus heteroclitus tolerate a broad range of environmental conditions including temperature, salinity, hypoxia and chemical pollutants. Strikingly, populations of Fundulus inhabit and have adapted to highly polluted Superfund sites that are contaminated with persistent toxic chemicals. These natural populations provide a foundation to discover critical gene pathways that have evolved in a complex natural environment in response to environmental stressors.ResultsWe used Fundulus cDNA arrays to compare metabolic gene expression patterns in the brains of individuals among nine populations: three independent, polluted Superfund populations and two genetically similar, reference populations for each Superfund population. We found that up to 17% of metabolic genes have evolved adaptive changes in gene expression in these Superfund populations. Among these genes, two (1.2%) show a conserved response among three polluted populations, suggesting common, independently evolved mechanisms for adaptation to environmental pollution in these natural populations.ConclusionSignificant differences among individuals between polluted and reference populations, statistical analyses indicating shared adaptive changes among the Superfund populations, and lack of reduction in gene expression variation suggest that common mechanisms of adaptive resistance to anthropogenic pollutants have evolved independently in multiple Fundulus populations. Among three independent, Superfund populations, two genes have a common response indicating that high selective pressures may favor specific responses.

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities, Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b5 content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23-2.1 nmol/min/mg and 0.5-11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with beta-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6 beta- and 16 beta-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.

Functional Genomics of Physiological Plasticity and Local Adaptation in Killifish

Evolutionary solutions to the physiological challenges of life in highly variable habitats can span the continuum from evolution of a cosmopolitan plastic phenotype to the evolution of locally adapted phenotypes. Killifish (Fundulus sp.) have evolved both highly plastic and locally adapted phenotypes within different selective contexts, providing a comparative system in which to explore the genomic underpinnings of physiological plasticity and adaptive variation. Importantly, extensive variation exists among populations and species for tolerance to a variety of stressors, and we exploit this variation in comparative studies to yield insights into the genomic basis of evolved phenotypic variation. Notably, species of Fundulus occupy the continuum of osmotic habitats from freshwater to marine and populations within Fundulus heteroclitus span far greater variation in pollution tolerance than across all species of fish. Here, we explore how transcriptome regulation underpins extreme physiological plasticity on osmotic shock and how genomic and transcriptomic variation is associated with locally evolved pollution tolerance. We show that F. heteroclitus quickly acclimate to extreme osmotic shock by mounting a dramatic rapid transcriptomic response including an early crisis control phase followed by a tissue remodeling phase involving many regulatory pathways. We also show that convergent evolution of locally adapted pollution tolerance involves complex patterns of gene expression and genome sequence variation, which is confounded with body-weight dependence for some genes. Similarly, exploiting the natural phenotypic variation associated with other established and emerging model organisms is likely to greatly accelerate the pace of discovery of the genomic basis of phenotypic variation.

RNA-Seq reveals complex genetic response to deepwater horizon oil release in Fundulus grandis

BackgroundThe release of oil resulting from the blowout of the Deepwater Horizon (DH) drilling platform was one of the largest in history discharging more than 189 million gallons of oil and subject to widespread application of oil dispersants. This event impacted a wide range of ecological habitats with a complex mix of pollutants whose biological impact is still not yet fully understood. To better understand the effects on a vertebrate genome, we studied gene expression in the salt marsh minnow Fundulus grandis, which is local to the northern coast of the Gulf of Mexico and is a sister species of the ecotoxicological model Fundulus heteroclitus. To assess genomic changes, we quantified mRNA expression using high throughput sequencing technologies (RNA-Seq) in F. grandis populations in the marshes and estuaries impacted by DH oil release. This application of RNA-Seq to a non-model, wild, and ecologically significant organism is an important evaluation of the technology to quickly assess similar events in the future.ResultsOur de novo assembly of RNA-Seq data produced a large set of sequences which included many duplicates and fragments. In many cases several of these could be associated with a common reference sequence using blast to query a reference database. This reduced the set of significant genes to 1,070 down-regulated and 1,251 up-regulated genes. These genes indicate a broad and complex genomic response to DH oil exposure including the expected AHR-mediated response and CYP genes. In addition a response to hypoxic conditions and an immune response are also indicated. Several genes in the choriogenin family were down-regulated in the exposed group; a response that is consistent with AH exposure. These analyses are in agreement with oligonucleotide-based microarray analyses, and describe only a subset of significant genes with aberrant regulation in the exposed set.ConclusionRNA-Seq may be successfully applied to feral and extremely polymorphic organisms that do not have an underlying genome sequence assembly to address timely environmental problems. Additionally, the observed changes in a large set of transcript expression levels are indicative of a complex response to the varied petroleum components to which the fish were exposed.

SNP identification, verification, and utility for population genetics in a non-model genus

BackgroundBy targeting SNPs contained in both coding and non-coding areas of the genome, we are able to identify genetic differences and characterize genome-wide patterns of variation among individuals, populations and species. We investigated the utility of 454 sequencing and MassARRAY genotyping for population genetics in natural populations of the teleost, Fundulus heteroclitus as well as closely related Fundulus species (F. grandis, F. majalis and F. similis).ResultsWe used 454 pyrosequencing and MassARRAY genotyping technology to identify and type 458 genome-wide SNPs and determine genetic differentiation within and between populations and species of Fundulus. Specifically, pyrosequencing identified 96 putative SNPs across coding and non-coding regions of the F. heteroclitus genome: 88.8% were verified as true SNPs with MassARRAY. Additionally, putative SNPs identified in F. heteroclitus EST sequences were verified in most (86.5%) F. heteroclitus individuals; fewer were genotyped in F. grandis (74.4%), F. majalis (72.9%), and F. similis (60.7%) individuals. SNPs were polymorphic and showed latitudinal clinal variation separating northern and southern populations and established isolation by distance in F. heteroclitus populations. In F. grandis, SNPs were less polymorphic but still established isolation by distance. Markers differentiated species and populations.ConclusionsIn total, these approaches were used to quickly determine differences within the Fundulus genome and provide markers for population genetic studies.